WO2023124185A1 - Off-the-shelf human umbilical cord derived mesenchymal stem cell, preparation method and application thereof - Google Patents

Abstract

The invention provides an off-the-shelf human umbilical cord derived mesenchymal stem cell, a preparation method thereof, and an application. The preparation method comprises the following steps: culturing an isolated umbilical cord tissue block to obtain a P0-generation cell; passaging the P0-generation cell twice to obtain a P2-generation cell and cryopreserving same; resuscitating the cryopreserved P2-generation cell, passaging twice to obtain a P4-generation cell and cryopreserving same; resuscitating the cryopreserved P4-generation cell, passaging once to obtain a P5-generation cell, and after sequential digestion, washing and resuspension of same, obtaining a human umbilical cord derived mesenchymal stem cell. Further, adopting a cryopreservation storage method, a human umbilical cord derived mesenchymal stem cell cryopreserved preparation is developed. The cryopreserved preparation is convenient to store, and after resuscitation, intravenous drug administration can be directly carried out, with no liquid change needed. Thus, the risk of possible pollution creation during medicine dispensing is avoided, facilitating clinical use. Further, the storage life of the cryopreserved preparation is long, and during the shelf life of same, multiple tests for preparation function and safety can be completed.

Description

A kind of spot type human umbilical cord-derived mesenchymal stem cells and its preparation method and application technical field

The invention belongs to the field of biotechnology, and in particular relates to a spot-type human umbilical cord-derived mesenchymal stem cell and a preparation method and application thereof.

Background technique

Mesenchymal stem cells (MSCs) are a group of multipotent adult stem cells derived from mesoderm, which have the potential of self-renewal, multi-directional differentiation, promotion of tissue and organ repair, and immune regulation. MSCs can be isolated and cultured from multiple tissues such as bone marrow, umbilical cord, placenta, fat, bone, dental pulp and endometrium, and MSC-like cells can also be differentiated from embryonic stem cells or pluripotent stem cells. MSC has low immunogenicity, and because it does not express costimulatory molecules such as CD40, CD80, and CD86, and major histocompatibility complex class II molecules, it is less likely to cause immune rejection. At the same time, because its regulatory effect on immune cells is not limited by major histocompatibility complex molecules, it can exert immune regulatory effects on both autologous and allogeneic immune cells. MSCs also have inflammatory chemotactic properties, and migrate to inflammatory sites by sensing inflammatory signals (cytokines, chemokine receptors, integrins, etc.), relying on direct cell-to-cell contact and/or paracrine effects to function, such as MSC can regulate the proliferation, differentiation and antibody production of abnormally activated T lymphocytes, B lymphocytes, natural killer cells, dendritic cells, etc. through direct contact between cells or secreting cytokines. A number of clinical studies It has been proved that MSC infusion can significantly reduce the secretion of proinflammatory cytokines, IFN-γ, IL-2, IL-12 and IL-17A in patients, and the secretion of IL-10 and TGF-β promoted by MSC can inhibit abnormally activated Th1 cells, restore Th1/Th2 balance, inhibit excessive proliferation of T cells, and inhibit the activity of cytotoxic CD8+ T lymphocytes through the NKG2D pathway. MSCs can also repolarize macrophages from a pro-inflammatory M1 phenotype to an anti-inflammatory M2 phenotype through the synergistic action of their own secreted IL-10 and TGF-β cytokines in an LPS-dependent manner. Studies have confirmed that after MSC treatment, the white blood cell count and neutrophil count of patients decreased to normal levels, and the counts of CD3+T cells, CD4+T cells and CD8+T cells also increased to normal levels.

At present, mesenchymal stem cells have been widely used clinically to treat a variety of diseases. The mesenchymal stem cell drugs or mesenchymal stem cell treatment technologies that have been approved in the world are as follows: 1. The autologous fat-derived medicine approved in South Korea in 2010 Queencell, the indication is subcutaneous tissue defect; 2. Cellgram-AMI, derived from autologous bone marrow, approved in South Korea in 2011, and the indication is acute myocardial infarction; 3. Cartistem, derived from allogeneic cord blood, approved in South Korea in 2012 , the indication is knee cartilage defect; 4. Cupistem, which is derived from autologous fat, was approved in South Korea in 2012, and the indication is Crohn's disease combined with anal fistula; 5. Prochymal, which is derived from allogeneic bone marrow, was approved in New Zealand in 2012 , the indication is acute graft-versus-host disease (children); 6. Neronata-R, derived from autologous bone marrow, was approved in South Korea in 2014, and the indication is amyotrophic lateral sclerosis; 7. It was approved in Canada in 2015 Prochymal derived from allogeneic bone marrow, indicated for acute graft-versus-host disease (children); 8. Temcell, derived from allogeneic bone marrow approved in Japan in 2015, indicated for acute graft-versus-host disease; 9. 2018 Alofisel, derived from allogeneic fat, approved in the EU, is indicated for Crohn's disease compound anal fistula; Stemirac, derived from allogeneic bone marrow, approved in Japan in 2018, indicated for spinal cord injury; 11, in India in 2020 The approved Stempeucel derived from allogeneic fat is indicated for severe limb ischemia; 12. The indication of Alofisel derived from allogeneic fat approved in Japan in 2021 is Crohn's disease combined with anal fistula. It can be seen that the stem cells in the current mesenchymal stem cell therapy technology are usually derived from bone marrow or fat, and no mesenchymal stem cell drug or treatment technology derived from umbilical cord has been approved for marketing. Compared with bone marrow or fat, umbilical cord-derived Mesenchymal stem cells have lower immunogenicity, can be used for allogeneic therapy, are easier to obtain, and are more suitable for large-scale production. At the same time, in the prior art, a culture system containing fetal bovine serum is usually selected, and exogenous serum is introduced, which poses a safety risk. Moreover, in the prior art, fresh preparations are usually used after resuscitated culture, and the biological activity of the cells is difficult to maintain for a long time, so it needs to be used or injected in time. These are the problems that the mesenchymal stem cell industry urgently needs to solve.

invention disclosure

The purpose of the present invention is to provide a preparation method and application of human umbilical cord-derived mesenchymal stem cells and their cryopreserved preparations.

In order to achieve the above object, the present invention firstly provides a preparation method of human umbilical cord-derived mesenchymal stem cells.

The preparation method of human umbilical cord-derived mesenchymal stem cells provided by the present invention comprises the following steps:

(1) culturing isolated umbilical cord tissue pieces, and obtaining P0 generation mesenchymal stem cells when the cell confluence of the mesenchymal stem cells climbing out from the umbilical cord tissue pieces is 40%-60%;

(2) After step (1) is completed, the P0 generation mesenchymal stem cells are subcultured until the cell fusion degree is 60%-80%, and the P1 generation mesenchymal stem cells are obtained;

(3) After completing step (2), subculture the P1-generation mesenchymal stem cells until the cell confluence is 60%-80%, and obtain P2-generation mesenchymal stem cells;

(4) After step (3) is completed, the P2 generation mesenchymal stem cells are cryopreserved to obtain frozen P2 generation seed bank cells;

(5) After completing step (4), the frozen P2 generation seed bank cells are recovered and subcultured, and cultured until the cell confluence is 60%-80%, to obtain P3 generation mesenchymal stem cells;

(6) After step (5) is completed, the P3 generation mesenchymal stem cells are subcultured until the cell confluence is 60%-80%, and the P4 generation mesenchymal stem cells are obtained;

(7) After step (6) is completed, the P4 generation mesenchymal stem cells are cryopreserved to obtain frozen P4 generation working bank cells;

(8) After completing step (7), the frozen P4 generation working bank cells are recovered and subcultured, and the P5 generation mesenchymal stem cells are obtained when the cell confluency is 60%-80%;

(9) After step (8), the P5 passage mesenchymal stem cells are digested, washed and resuspended in sequence to obtain the human umbilical cord-derived mesenchymal stem cells.

In the above-mentioned preparation method of human umbilical cord-derived mesenchymal stem cells, the following steps are also included before the step (1): take the isolated umbilical cord and sterilize it to obtain a sterilized umbilical cord; then clean the sterilized umbilical cord and cut it into 1 -2cm small pieces to obtain small pieces of umbilical cord; then clean the small piece of umbilical cord and cut it into 1-2mm ³ tissue pieces.

In the step (1), the isolated umbilical cord tissue pieces are cultured with complete medium of fetal bovine serum. The culture method may specifically include the following steps: adding complete fetal bovine serum medium to the cell culture flask containing the umbilical cord tissue block, and inverting it to 5% CO ₂ and culturing at 37°C; after 4 hours of culturing, the cell culture flask Place in 5% CO ₂ , saturated humidity, and 37°C to continue culturing; after 24 hours of culture, add fetal bovine serum complete medium to the cell culture flask, and place in 5% CO ₂ , saturated humidity, 37°C Continue to culture; after 7 days of umbilical cord tissue patch culture, discard the culture medium, add fetal calf serum complete medium to the cell culture flask, and place it in 5% CO ₂ , saturated humidity, and 37°C to continue the culture.

In the step (2), the fetal bovine serum complete medium is used for subculture. The method for subculture (P0 to P1) may specifically include the following steps: absorb all the culture medium, add sodium chloride injection (0.9%) to the cell culture flask to wash once, then add TrypLE for digestion, and wait until the cells are completely After suspension, add sodium chloride injection (0.9%) to stop digestion, and move the cell suspension to a centrifuge tube; wash each cell culture bottle with sodium chloride injection (0.9%), and wash the obtained cell suspension Transfer them together into a centrifuge tube; centrifuge the centrifuge tube (300g for 8 minutes), aspirate and discard the supernatant, resuspend the cells with complete fetal bovine serum medium equilibrated to room temperature, mix well and take the cell suspension for counting, calculate The total number of harvested P0 generation cells; inoculated in cell culture flasks at a density of 18,000-20,000 cells/cm ² , supplemented each bottle with complete fetal bovine serum medium to the standard volume, marked and placed in 5% CO ₂ , The cultivation was continued under the conditions of saturated humidity and 37°C.

In the step (3), the fetal bovine serum complete medium is used for subculture. The method for subculture (P1 to P2) may specifically include the following steps: absorb and discard all the medium, add sodium chloride injection (0.9%) to the cell culture flask to wash once, then add TrypLE for digestion, and wait until the cells are completely After suspension, add sodium chloride injection (0.9%) to stop digestion, and move the cell suspension to a centrifuge tube; wash each cell culture bottle with sodium chloride injection (0.9%), and wash the obtained cell suspension Transfer them together into a centrifuge tube; centrifuge the centrifuge tube (300g for 8 minutes), aspirate and discard the supernatant, resuspend the cells with complete fetal bovine serum medium equilibrated to room temperature, mix well and take the cell suspension for counting, calculate The total number of harvested P1 generation cells; inoculated in cell culture flasks at a density of 18,000-20,000 cells/cm ² , supplemented each bottle with complete fetal bovine serum medium to the standard volume, marked and placed in 5% CO ₂ , The cultivation was continued under the conditions of saturated humidity and 37°C.

In the step (4), the method for freezing the cells may specifically include the following steps: absorb and discard all the culture fluid, add sodium chloride injection (0.9%) to the cell culture flask to wash once, and then add TrypLE for digestion After the cells are completely suspended, add sodium chloride injection (0.9%) to stop digestion, and move the cell suspension to a centrifuge tube; wash each culture bottle with sodium chloride injection (0.9%), and wash the obtained Transfer the cell suspension into a centrifuge tube together; centrifuge the centrifuge tube (300g for 8 minutes), discard the supernatant, resuspend the cells with freezing solution A, mix well, take the cell suspension for counting, and calculate the harvested P2 generation The total number of cells; according to the counting results, add the required volume of cryopreservation solution A to make a cell suspension, so that the cell concentration is 3E6 cells/mL, and add the cell suspension into a cryopreservation tube, and put it into a gradient cooling box (4 °C pre-cooling) and then placed in a -80 °C low-temperature refrigerator for freezing, and transferred to a liquid nitrogen storage tank within 1 month. Further, the cryopreservation solution A consists of fetal bovine serum and dimethyl sulfoxide. Furthermore, the volume ratio of the fetal bovine serum and the dimethyl sulfoxide may be 9:1.

In the step (5), the recovery method may specifically include the following steps: putting the cryopreservation tube containing the P2 generation seed bank cells into a 37°C water bath until the frozen cells are completely thawed; Transfer the cell suspension in the storage tube to a centrifuge tube filled with serum-free complete medium, mix well and centrifuge (300g for 5 minutes), discard the supernatant, and resuspend the cells with serum-free complete medium equilibrated to room temperature. Serum-free complete medium was used for subculture. The method for subculture (P2 to P3) may specifically include the following steps: inoculate cell culture bottles at a density of 8,000-9,000 cells/cm ² , add serum-free complete medium to a standard volume for each bottle, and prepare After labeling, culture was continued under the conditions of 5% CO ₂ , saturated humidity, and 37°C.

In the step (6), the serum-free complete medium is used for subculture. The method for subculture (P3 to P4) may specifically include the following steps: absorb and discard all the medium, add sodium chloride injection (0.9%) to the cell culture flask to wash once, then add TrypLE for digestion, and wait until the cells are completely After suspension, add sodium chloride injection (0.9%) to stop digestion, and move the cell suspension to a centrifuge tube; wash each cell culture bottle with sodium chloride injection (0.9%), and wash the obtained cell suspension Transfer them together into a centrifuge tube; centrifuge the centrifuge tube (300g for 8 minutes), discard the supernatant, resuspend the cells with serum-free complete medium equilibrated to room temperature, mix well and take the cell suspension for counting, and calculate the harvest The total number of cells in the P3 generation; inoculate in cell culture flasks at a density of 6,000-8,000 cells/cm ² , add complete serum-free medium to the standard volume for each bottle, mark it and place it in 5% CO ₂ and saturated humidity , Continue culturing at 37°C.

In the step (7), the method for freezing the cells may specifically include the following steps: absorb and discard all the culture fluid, add sodium chloride injection (0.9%) to the cell culture flask to wash once, and then add TrypLE for digestion After the cells are completely suspended, add sodium chloride injection (0.9%) to stop digestion, and move the cell suspension to a centrifuge tube; wash each culture bottle with sodium chloride injection (0.9%), and wash the obtained Transfer the cell suspension into a centrifuge tube together; centrifuge the centrifuge tube (300g for 8 minutes), aspirate and discard the supernatant, and resuspend the cells in freezing solution B to make the cell density 8E6 cells/mL; Put it into a cryopreservation tube, put it into a gradient cooling box (pre-cooled at 4°C), freeze it in a low-temperature refrigerator at -80°C, and transfer it to a liquid nitrogen storage tank within 1 month. Further, the cryopreservation solution B consists of serum-free medium base, dimethyl sulfoxide and human serum albumin solution. Further, the volume ratio of the serum-free medium base, the dimethyl sulfoxide and the human albumin solution (0.2 g/mL) may be 7:2:1.

In the step (8), the recovery method may specifically include the following steps: putting the cryopreservation tube containing the P4 generation working library cells into a 37°C water bath until the frozen cells are completely thawed; Transfer the cell suspension in the storage tube to a centrifuge tube filled with serum-free complete medium, mix well and centrifuge (300g for 5 minutes), discard the supernatant, and resuspend the cells with serum-free complete medium equilibrated to room temperature. Serum-free complete medium was used for subculture. The method for subculture (P4 to P5) may specifically include the following steps: inoculate cell culture bottles at a density of 8,000-11,000 cells/cm ² , add serum-free complete medium to a standard volume for each bottle, and prepare After labeling, culture was continued under the conditions of 5% CO ₂ , saturated humidity, and 37°C.

The step (9) may specifically include the following steps: suck and discard all the culture fluid, add sodium chloride injection (0.9%) to the cell culture bottle to wash once, then add TrypLE for digestion, and then add chlorine after the cells are completely suspended. Sodium chloride injection (0.9%) terminates the digestion, and the cell suspension is moved to a centrifuge tube; each culture bottle is cleaned with sodium chloride injection (0.9%), and the cell suspension obtained by cleaning is moved into a centrifuge tube Centrifuge the centrifuge tube (300g for 8 minutes), suck and discard the supernatant, wash the cells with sodium chloride injection (0.9%) containing human albumin (0.1%), and centrifuge (300g for 8 minutes); repeat the centrifugation Washing step: when washing for the third time, resuspend the cells with sodium chloride injection (0.9%) containing human serum albumin (0.1%) to obtain a stock solution of human umbilical cord-derived mesenchymal stem cells, which contains said Human umbilical cord-derived mesenchymal stem cells.

In the above method, the fetal bovine serum complete medium consists of DMEM/F12 basal medium and fetal bovine serum. The volume ratio of the DMEM/F12 basal medium and the fetal bovine serum can be 9:1.

The serum-free complete medium is composed of a serum-free medium base for mesenchymal stem cells and a serum-free medium supplement for mesenchymal stem cells. The volume ratio of the serum-free medium base for mesenchymal stem cells and the serum-free medium supplement for mesenchymal stem cells may be 100:1.

In order to achieve the above purpose, the present invention further provides human umbilical cord-derived mesenchymal stem cells prepared according to the above method.

In order to achieve the above object, the present invention also provides the application of the above-mentioned human umbilical cord-derived mesenchymal stem cells in the preparation of cryopreserved preparations of human umbilical cord-derived mesenchymal stem cells.

In order to achieve the above purpose, the present invention also provides a cryopreserved preparation of human umbilical cord-derived mesenchymal stem cells.

The cryopreserved preparation of human umbilical cord-derived mesenchymal stem cells provided by the present invention includes the above-mentioned human umbilical cord-derived mesenchymal stem cells and a cell cryoprotectant.

Further, the cell cryoprotectant is composed of compound electrolyte solution, dimethyl sulfoxide, dextran 40 sodium chloride injection and human serum albumin solution (0.2g/mL).

The concentration of the human umbilical cord-derived mesenchymal stem cells in the frozen preparation may be 2.5×10 ⁶ -1×10 ⁷ cells/mL, specifically 2.5×10 ⁶ cells/mL, 5×10 ⁶ cells/mL, 1×10 ⁷ cells/mL.

Further, the volume ratio of the compound electrolyte solution, the dimethyl sulfoxide, the dextran 40 sodium chloride injection and the human serum albumin solution (0.2g/mL) can be (70-80 ):(5-10):(5-10):(5-10) or 70:(5-10):(5-10):(5-10) or 80:(5-10):(5 -10): (5-10) or (70-80): 5: (5-10): (5-10) or (70-80): 10: (5-10): (5-10) or (70-80): (5-10): 5: (5-10) or (70-80): (5-10): 10: (5-10) or (70-80): (5-10 ):(5-10):5 or (70-80):(5-10):(5-10):10.

In a specific embodiment of the present invention, the volume ratio of the compound electrolyte solution, the dimethyl sulfoxide, the dextran 40 sodium chloride injection and the human albumin solution (0.2g/mL) is 80:5:10:5 or 70:10:10:10 or 80:5:5:10 or 80:10:5:5.

In order to achieve the above object, the present invention also provides a preparation method for the above cryopreserved preparation of human umbilical cord-derived mesenchymal stem cells.

The preparation method of the human umbilical cord-derived mesenchymal stem cell cryopreservation preparation provided by the present invention comprises the following steps: resuspending the above-mentioned human umbilical cord-derived mesenchymal stem cells with a cell cryoprotectant, and then cooling the obtained cell suspension to obtain the obtained Describe human umbilical cord-derived mesenchymal stem cell cryopreservation preparation.

Further, the procedure of the cooling treatment is as follows:

Step 1: Initial temperature to 4°C;

Step 2: 1°C/min down to -5°C;

Step 3: 20°C/min until the cavity temperature is -40°C;

Step 4: 10°C/min until the cavity temperature is -20°C;

Step 5: 2°C/min until the sample drops to -40°C;

Step 6: 10°C/min until the sample drops to -80°C;

Step 7: End.

In order to achieve the above purpose, the present invention also provides a new application of the above-mentioned human umbilical cord-derived mesenchymal stem cells or the above-mentioned cryopreserved preparation of human umbilical cord-derived mesenchymal stem cells or the cryopreserved preparation of human umbilical cord-derived mesenchymal stem cells prepared according to the above method .

The present invention provides the above-mentioned human umbilical cord-derived mesenchymal stem cells or the above-mentioned cryopreserved preparations of human umbilical cord-derived mesenchymal stem cells or the cryopreserved preparations of human umbilical cord-derived mesenchymal stem cells prepared according to the above method in any of the following N1)-N10) Types of applications:

N1) Preparation of products for the prevention and/or treatment of diseases caused by novel coronaviruses;

N2) Preparation of products for preventing and/or treating idiopathic pulmonary interstitial fibrosis;

N3) Preparation of products for preventing and/or treating acute lung injury;

N4) Preparation of products for preventing and/or treating liver fibrosis;

N5) Preparation of products for preventing and/or treating Crohn's disease;

N6) Prevention and/or treatment of diseases caused by novel coronaviruses;

N7) prevention and/or treatment of idiopathic pulmonary interstitial fibrosis;

N8) prevention and/or treatment of acute lung injury;

N9) prevention and/or treatment of liver fibrosis;

N10) Prevention and/or treatment of Crohn's disease.

In order to achieve the above object, the present invention also provides a product, the active ingredient of which is the above-mentioned human umbilical cord-derived mesenchymal stem cells or the above-mentioned cryopreserved preparation of human umbilical cord-derived mesenchymal stem cells or the human umbilical cord prepared according to the above method Source mesenchymal stem cell cryopreservation preparation; the function of the product is any one of the following M1)-M5):

M1) Prevention and/or treatment of diseases caused by novel coronavirus;

M2) prevention and/or treatment of idiopathic pulmonary interstitial fibrosis;

M3) prevention and/or treatment of acute lung injury;

M4) prevention and/or treatment of liver fibrosis;

M5) Prevention and/or treatment of Crohn's disease.

In order to achieve the above purpose, the present invention finally provides a method for treating diseases caused by novel coronavirus or idiopathic pulmonary interstitial fibrosis or acute lung injury or liver fibrosis or Crohn's disease.

The method for treating diseases caused by novel coronavirus or idiopathic pulmonary interstitial fibrosis or acute lung injury or liver fibrosis or Crohn's disease provided by the present invention comprises the following steps: Patients with chronic pulmonary interstitial fibrosis or acute lung injury or patients with liver fibrosis or Crohn's disease were administered the above-mentioned human umbilical cord-derived mesenchymal stem cells or the above-mentioned cryopreserved preparations of human umbilical cord-derived mesenchymal stem cells or prepared according to the above method The cryopreserved preparation of human umbilical cord-derived mesenchymal stem cells enables the patient to be treated.

In any of the above-mentioned applications or products, the prevention and/or treatment of diseases caused by novel coronaviruses is reflected in reducing the viral load of the lungs and/or improving the inflammatory infiltration around small blood vessels and/or improving the inflammatory infiltration around bronchioles And/or improve lung bronchiole epithelial cell degeneration.

The prevention and/or treatment of idiopathic pulmonary interstitial fibrosis is embodied by improving the lung function damage caused by fibrosis and/or inhibiting the expression of pro-fibrosis factors (such as HYP, MMP-2, TIMP-1) and/or Improve collagen deposition in lung tissue and/or improve pulmonary interstitial or tracheal or perivascular or alveolar inflammatory infiltration and fibrosis.

The prevention and/or treatment of acute lung injury is manifested by reducing lung water content and/or improving pulmonary edema and/or improving acute respiratory distress syndrome (ARDS) and/or improving lung inflammation and/or improving lung tissue inflammatory infiltration And/or improve the lesion degree of lymphoid tissue hyperplasia.

The prevention and/or treatment of liver fibrosis is reflected in improving the four indexes of liver fibrosis and/or improving liver structure and/or improving liver collagen deposition.

The prevention and/or treatment of Crohn's disease is embodied by reducing the area of colonic ulcers and/or reducing the incidence of ulcers and/or alleviating colonic damage.

In any of the above applications or products, the product can be a drug.

In any of the aforementioned applications or product methods, the disease caused by the novel coronavirus is novel coronavirus pneumonia (COVID-19). The novel coronavirus is SARS-CoV-2, specifically SARS-CoV-2 (HRB26) strain.

Description of drawings

Figure 1 is a morphological diagram of human umbilical cord-derived mesenchymal stem cells.

Figure 2 is a staining diagram of the differentiation of human umbilical cord-derived mesenchymal stem cells. From top to bottom, after 21 days of osteogenic induction and differentiation, Alizarin Red-S staining was positive; after 14 days of adipogenic induction and differentiation, Oil Red-O staining was positive; after 21 days of chondrogenic differentiation, after paraffin section Blue staining is positive.

Figure 3 shows the viability of cells after recovery.

Fig. 4 is a graph showing the expression level of IDO gene mRNA in human umbilical cord-derived mesenchymal stem cells.

Fig. 5 is a graph showing the expression level of CCL2 gene mRNA in human umbilical cord-derived mesenchymal stem cells.

Fig. 6 is a graph showing the expression level of CXCL10 gene mRNA in human umbilical cord-derived mesenchymal stem cells.

Figure 7 is the effect of the test product on the pathological changes of the lung tissue of the BLM-induced pulmonary fibrosis model rat. Note: HE staining, 100×. a. Normal control group, no obvious lesions were seen under the microscope in the right upper lobe of the lung; b. Model control group, alveolitis cell infiltration (obvious) and interstitial/alveolar fibrosis (obvious) were seen in the right upper lobe of the lung; c. Low-dose C1 group , alveolitis cell infiltration (moderate) and interstitial/alveolar fibrosis (moderate) can be seen in the right upper lobe of the lung; d. e. In the C1 high-dose group, alveolar inflammatory cell infiltration (mild) and interstitial/alveolar fibrosis (mild) were seen under the microscope in the right upper lobe of the lung.

Figure 8 is the effect of the test product on the lung histopathology of ALI model rats. Note: HE staining, 40×. a. In the normal control group, no obvious abnormal changes were found in the right lung. b. Model control group, right lung interstitium/alveolitis cell infiltration and bronchi-associated lymphoid tissue hyperplasia. c.C1 low-dose group, right lung interstitium/alveolitis cell infiltration and broncho-associated lymphoid tissue hyperplasia. d. The right lung interstitium/alveolitis cell infiltration and broncho-associated lymphoid tissue hyperplasia in the middle dose group of C1. e.C1 high-dose group, right lung interstitium/alveolitis cell infiltration and broncho-associated lymphoid tissue hyperplasia.

The best way to practice the invention

The present invention will be further described in detail below in conjunction with specific embodiments, and the given examples are only for clarifying the present invention, not for limiting the scope of the present invention. The examples provided below can be used as a guideline for those skilled in the art to make further improvements, and are not intended to limit the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Fetal bovine serum complete medium formula: 10% fetal bovine serum (Biological Industries (BI), catalog number: 04-001-1ACS) + 90% DMEM/F12 basal medium (Biological Industries (BI), catalog number: 04-172- 1ACS).

Serum-free complete medium formula: For every 500mL mesenchymal stem cell serum-free medium base (Youkang Biotechnology, product number: NC0103), add 5mL mesenchymal stem cell serum-free medium supplement (Youkang Biotechnology, product number: NC0103.S).

Chondrogenic Induction Differentiation Complete Medium: Biological Industries (BI), Cat. No.: 05-220-1B.

Osteogenic induction medium: Biological Industries (BI), Cat. No.: 05-440-1B.

Adipogenic Induction Medium: Biological Industries (BI), Cat. No.: 05-330-1B.

TrypLE: Thermo Fisher (GIBCO), Cat. No. 12563-029.

Mouse IgG1-FITC: BD Biosciences, Cat. No. 555748.

antiCD19-FITC: BD Biosciences, Cat. No.: 555412.

antiCD34-FITC: BD Biosciences, Cat. No.: 555821.

Mouse IgG1-PE: BD Biosciences, Cat. No. 554680.

antiCD11b-PE: BD Biosciences, Cat. No.: 555388.

antiCD73-PE: BD Biosciences, Cat. No. 550257.

antiCD90-PE: BD Biosciences, Cat. No.: 555596.

antiCD45-PE: BD Biosciences, Cat. No.: 555483.

antiCD105-PE: BD Biosciences, Cat. No.: 560839.

antiHLA-DR-PE: BD Biosciences, Cat. No.: 555812.

Compound electrolyte injection: Shanghai Baite Medical Supplies Co., Ltd., article number: Guoyao Zhunzi H20000475.

Dimethyl sulfoxide (DMSO): Hunan Jiudian Pharmaceutical Co., Ltd., article number: Xiangshiyaofuzhunzi F20090010.

Dextran 40 Sodium Chloride Injection: Shijiazhuang No. 4 Medicine Co., Ltd., article number: Guoyao Zhunzi H13022493.

Human serum albumin solution (0.2g/mL): Switzerland Jet Behring Biological Products Co., Ltd.

IFN-γ: Inshore Organisms, Cat. No.: C014.

0.9% Sodium Chloride Injection: Shijiazhuang No.4 Medicine Co., Ltd., product number: Guoyao Zhunzi H13023201.

Example 1. Preparation and identification of human umbilical cord-derived mesenchymal stem cells

(1) Preparation of human umbilical cord-derived mesenchymal stem cells

1. Isolation of human umbilical cord-derived mesenchymal stem cells

1. Take the isolated umbilical cords of full-term newborns without congenital diseases (the newborns have no infectious diseases such as hepatitis, syphilis, AIDS, etc., and the mothers and their families have informed consent for the umbilical cords to be used in experimental research) and put them into a 50mL Sterilize the beaker with 75% ethanol for 10s. Then rinse with 30mL 0.9% Sodium Chloride Injection (Shijiazhuang No.4 Medicine Co., Ltd., National Drug Approval H13023201) three times.

2. After completing step 1, add 50mL of 0.9% sodium chloride injection into the kidney-shaped dish, then put the umbilical cord into the kidney-shaped dish to remove surface blood stains, then cut the umbilical cord into 1-2cm pieces with surgical scissors, and then drain blood, discard the waste.

3. After completing step 2, put the small section of umbilical cord into a beaker filled with 30mL 0.9% sodium chloride injection, rinse it three times repeatedly, and then place the cleaned small section of umbilical cord in a dry glass beaker under clean conditions. Cut the umbilical cord into small pieces of about 1-2 mm ³ with surgical scissors, and inoculate them evenly in T75 cell culture flasks.

4. After completing step 3, add 4 mL of complete fetal bovine serum medium into the T75 cell culture flask, and place it upside down in a 5% CO ₂ , 37° C. incubator. After 4 hours, the culture bottle was placed in a 5% CO ₂ , saturated humidity, and the culture was continued in a 37° C. incubator. After 24 hours, each T75 culture flask was supplemented with 11 mL of complete fetal bovine serum medium, placed in 5% CO ₂ , saturated humidity, and continued to culture in a 37° C. incubator. After the umbilical cord tissue block was cultured for 7 days, the culture solution was discarded, and 15 mL of complete fetal bovine serum medium was added to each T75 culture bottle, placed in 5% CO ₂ , saturated humidity, and continued to culture in a 37°C incubator. During culture, mesenchymal stem cells crawled out of the umbilical cord tissue mass.

2. Seed bank establishment (P0-P2)

1. When the umbilical cord tissue block is cultured 9-13 days after the cell fusion degree of the whole bottle reaches 40%-60%, the P0 generation mesenchymal stem cells are obtained, and then the P0-P1 subculture is carried out. The specific method is as follows: absorb Discard all the medium, add 5mL 0.9% sodium chloride injection to each T75 culture bottle to wash once, then add 2.5mL TrypLE, digest for about 2 minutes, after the cells are completely suspended, add 5mL 0.9% sodium chloride injection to stop Digest and transfer the cell suspension to a centrifuge tube. Wash each culture bottle with 5 mL of 0.9% sodium chloride injection, and transfer the cell suspension obtained by washing into a centrifuge tube. Centrifuge the centrifuge tube at 300g for 8 minutes, discard the supernatant after centrifugation, resuspend the cells with complete fetal bovine serum medium equilibrated to room temperature, mix well, take the cell suspension for counting, and calculate the total number of harvested P0 generation cells. Seed in culture flasks at a density of 18,000-20,000 cells/cm ² . Each bottle was supplemented with fetal bovine serum complete medium to the standard volume (T75: 15mL; T175: 35mL), marked and placed in 5% CO ₂ , saturated humidity, and continued cultivation in a 37°C incubator.

2. After step 1 is completed, when the cell confluence of the whole bottle reaches 60%-80% 48-72 hours after inoculation, the P1 generation mesenchymal stem cells are obtained, and then P1-P2 subculture is carried out. The specific method is as follows: discard all the culture medium, add 0.9% sodium chloride injection (T75: 5mL; T175: 10mL) to each bottle to wash once, then add TrypLE (T75: 2.5mL; T175: 5mL) to digest for about 2 minutes, After the cells were completely suspended, 0.9% sodium chloride injection (T75: 5 mL; T175: 10 mL) was added to stop the digestion, and the cell suspension was transferred to a 250 mL centrifuge tube. Wash each culture flask with 0.9% sodium chloride injection (T75: 5 mL; T175: 10 mL), and transfer the washed cell suspension into a centrifuge tube. Centrifuge the centrifuge tube at 300g for 8 minutes, discard the supernatant after centrifugation, resuspend the cells with complete fetal bovine serum medium equilibrated to room temperature, mix well, take the cell suspension for counting, and calculate the total number of harvested P1 generation cells. Inoculate in culture flasks at a density of 18,000-20,000 cells/cm ² , supplement each bottle with complete fetal bovine serum medium to a standard volume (T175: 35mL; T525: 105mL), mark and place in 5% CO ₂ , saturated humidity, and continue to cultivate in a 37°C incubator.

3. After step 2 is completed, when the cell confluence of the whole bottle reaches 60%-80% 48-72 hours after inoculation, the P2 generation mesenchymal stem cells are obtained, and then the P2 generation seed bank cells are frozen. The specific method is as follows: absorb and discard all the culture medium, add 0.9% sodium chloride injection (T175: 10mL; T525: 30mL) to each bottle to wash once, then add TrypLE (T175: 5mL; T525: 15mL) to digest for about 2 minutes, wait for After the cells were completely suspended, 0.9% sodium chloride injection (T175: 10 mL; T525: 30 mL) was added to terminate the digestion, and the cell suspension was transferred to a 250 mL centrifuge tube. Wash each culture flask with 0.9% sodium chloride injection (T175: 10 mL; T525: 30 mL), and transfer the washed cell suspension into a centrifuge tube. Centrifuge the tube at 300g for 8 minutes. After centrifugation, the supernatant was discarded, and the cells were resuspended in the cryopreservation solution (fetal bovine serum: DMSO = 9:1 (volume ratio)). After mixing, the cell suspension was taken for counting, and the total number of harvested P2 generation cells was calculated. According to the counting results, add the required volume of cryopreservation solution to make a cell suspension, adjust the cell concentration to about 3E6 cells/mL, and add the cell suspension to the cryopreservation tube, 1.5 mL/tube. Put it into a pre-cooled gradient cooling box at 4°C, place it in a -80°C low-temperature refrigerator, and freeze it. Transfer to a liquid nitrogen storage tank within 1 month.

3. Work library establishment (P2-P4)

1. Take cryopreserved P2 generation cells for recovery. The specific method is as follows: put the cryopreservation tube containing the P2 generation cells into a 37°C water bath, and randomly check the thawing situation in 1-3 minutes until the frozen cells are completely thawed.

2. After completing step 1, transfer the cell suspension in the cryopreservation tube to a centrifuge tube filled with complete serum-free medium, mix well and centrifuge at 300g for 5 minutes. Resuspend cells in complete serum-free medium.

3. After completing step 2, subculture P2-P3. The specific method is as follows: inoculate culture flasks at a density of 8,000-9,000 cells/cm ² , add serum-free complete medium to each flask to a standard volume (T175: 35 mL; T525: 105 mL), mark and place in 5 %CO ₂ , saturated humidity, 37°C incubator to continue culturing.

4. After step 3 is completed, when the degree of cell confluence reaches 60%-80% 48-72 hours after inoculation, the P3 generation mesenchymal stem cells are obtained, and then P3-P4 subculture is carried out. The specific method is as follows: absorb and discard all the culture medium, add 0.9% sodium chloride injection (T175: 10mL; T525: 30mL) to each bottle to wash once, then add TrypLE (T175: 5mL; T525: 15mL) to digest for about 2 minutes, wait for After the cells were completely suspended, 0.9% sodium chloride injection (T175: 10 mL; T525: 30 mL) was added to terminate the digestion, and the cell suspension was transferred to a 250 mL centrifuge tube. Wash each culture flask with 0.9% sodium chloride injection (T175: 10 mL; T525: 30 mL), and transfer the washed cell suspension into a centrifuge tube. Centrifuge the centrifuge tube at 300g for 8 minutes, discard the supernatant after centrifugation, resuspend the cells with complete serum-free medium equilibrated to room temperature, mix well, take the cell suspension for counting, and calculate the total number of harvested P3 generation cells. Inoculate in culture flasks at a density of 6,000-8,000 cells/cm ² , add complete serum-free medium to the standard volume (T525: 105mL; T875: 175mL) in each bottle, mark and place in 5% CO ₂ , Saturated humidity, 37 ℃ incubator to continue cultivation.

5. After step 4 is completed, when the cell confluence reaches 60%-80% 48-72 hours after inoculation, the P4 passage mesenchymal stem cells are obtained, and then the P4 passage working bank cells are frozen. The specific method is as follows: absorb and discard all the culture medium, add 0.9% sodium chloride injection (T175: 10mL; T525: 30mL) to each bottle to wash once, then add TrypLE (T175: 5mL; T525: 15mL) to digest for about 2 minutes, wait for After the cells were completely suspended, 0.9% sodium chloride injection (T175: 10 mL; T525: 30 mL) was added to terminate the digestion, and the cell suspension was transferred to a 250 mL centrifuge tube. Wash each culture flask with 0.9% sodium chloride injection (T175: 10 mL; T525: 30 mL), and transfer the washed cell suspension into a centrifuge tube. Centrifuge the centrifuge tube at 300g for 8 minutes, discard the supernatant after centrifugation, and use the freezing solution (serum-free medium base: DMSO: human albumin solution (0.2g/mL) = 7:2:1 (volume ratio)) Resuspend the cells, mix well, take the cell suspension for counting, and calculate the total number of harvested P4 generation cells. According to the counting results, add the required volume of cryopreservation solution to make a cell suspension, adjust the cell concentration to 8E6 cells/mL, and add the cell suspension to the cryopreservation tube, 1.5 mL/tube. Put it into a pre-cooled gradient cooling box at 4°C, place it in a -80°C low-temperature refrigerator, and freeze it. Transfer to a liquid nitrogen storage tank within 1 month.

4. Cell stock solution preparation (P4-P5)

1. Take the cryopreserved P4 cells for recovery, the specific method is as follows: put the cryopreservation tube containing the P4 cells in a 37°C water bath, and randomly check the thawing situation in 1-3 minutes until the frozen cells are completely melt.

2. After completing step 1, transfer the cell suspension in the cryopreservation tube to a centrifuge tube filled with complete serum-free medium, mix well and centrifuge at 300g for 5 minutes. Resuspend cells in complete serum-free medium.

3. After completing step 2, subculture P4-P5. The specific method is as follows: inoculate in culture flasks at a density of 8,000-11,000 cells/cm ² , add complete serum-free medium to each bottle to a standard volume (T525: 105 mL), place in 5% CO ₂ after marking, Saturated humidity, 37 ℃ incubator to continue cultivation.

4. After step 3 is completed, when the degree of cell confluence reaches 60%-80% 48-72 hours after inoculation (the cell morphology is shown in Figure 1), the P5 generation mesenchymal stem cells are obtained, and then the stock solution is prepared. The specific method is as follows: Discard the culture medium in all culture bottles. Add 0.9% sodium chloride injection (T525: 30mL) to each culture flask to wash once, then add TrypLE (T525: 15mL) to digest for about 2 minutes, and then add 0.9% sodium chloride injection (T525 : 30mL) to terminate the digestion, and transfer the cell suspension to a 250mL centrifuge tube. Wash each culture flask with 0.9% sodium chloride injection (T525: 30 mL), transfer the cell suspension obtained from the washing into a centrifuge tube, and centrifuge at 300 g for 8 minutes. After centrifugation, the supernatant was discarded, and the cells were washed with 0.9% sodium chloride injection containing 0.1% human serum albumin, and centrifuged at 300g for 8 minutes. Repeat the centrifugation wash step. At the time of the third washing, the cells were resuspended with 0.9% sodium chloride injection containing 0.1% human serum albumin to obtain the stock solution of human umbilical cord-derived mesenchymal stem cells.

According to the above method, three umbilical cords from different sources (respectively numbered Y200001, Y200003, and Y210001) were used as raw materials to prepare stem cell stock solutions. The umbilical cords numbered Y200001 and Y200003 were obtained from Hubei Provincial People’s Hospital, and the umbilical cord numbered Y210001 was obtained from Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology.

(2) Identification of human umbilical cord-derived mesenchymal stem cells in the stock solution

1. Differentiation identification of human umbilical cord-derived mesenchymal stem cells in stock solution

1. Taking stem cell stocks from 3 different umbilical cord sources (numbers Y200001, Y200003, and Y210001) as an example, add 1 mL of cells with a concentration of 1-2E6/mL into a 15 mL centrifuge tube, and centrifuge at 300 g for 5 min. Then add 1 mL of complete chondrogenic differentiation medium to resuspend the cells, centrifuge at 200 g for 5 min at room temperature, place them in a 5% CO ₂ incubator at 37°C, and replace with complete chondrogenic differentiation medium every 3 days. After continuous induction for 21 days, the chondrocytes were fixed in 4% paraformaldehyde, embedded in paraffin and sectioned, finally stained with Alcian blue.

2. After step 1 is completed, cells are seeded in a well plate at a density of 8000 cells/mL, cultured in a 37°C, 5% _CO2 incubator, and divided into the following two groups for treatment:

Osteogenic induction group: when the cell fusion rate reaches 80%, discard the medium, add osteogenic induction medium, change fresh osteogenic induction medium every 3 days, culture for 21 days, and stain with Alizarin Red-S.

Adipogenic induction group: when the cell fusion rate reaches 100%, discard the medium, add adipogenic induction medium, and replace with fresh adipogenic induction medium every 2 days. Cultured for 14 days and stained with Oil Red O.

The result is shown in Figure 2. The results show that: the human umbilical cord-derived mesenchymal stem cells prepared by the method of the present invention have good differentiation performance and meet the characteristics of stem cells.

2. Identification of surface markers of human umbilical cord-derived mesenchymal stem cells in the stock solution

Taking stem cell stocks from three different umbilical cord sources (numbered Y200001, Y200003, and Y210001) as examples, the cell concentration was adjusted to 2×10 ⁶ /mL. Take the flow tube and add Mouse IgG1-FITC, antiCD19-FITC, antiCD34-FITC, Mouse IgG1-PE, antiCD11b-PE, antiCD73-PE, antiCD90-PE, antiCD45-PE, antiCD105-PE, antiHLA-DR- PE. The amount of antibody added was 5 μL, and 100 μL of the cell suspension to be tested was added respectively, shaken and mixed, and incubated at room temperature in the dark for 20 minutes. Add 2mL 1×PBS to each tube for washing, and centrifuge at 1200rpm for 5min. Discard the supernatant, add 200 μL 1×PBS to resuspend, mix well, and then use BD FACSCalibur flow cytometer to detect 10,000 cells per sample.

The results are shown in Table 1. The results show that the surface expression of CD73, CD90 and CD105 on the surface of mesenchymal stem cells prepared by the method of the present invention is greater than 95%, which is positive; CD34, CD45, CD11b, CD19 and HLA-DR are all less than 2%, which is negative, and after cryopreservation Cell surface markers did not change, consistent with the characteristics of stem cell surface markers.

Table 1. Detection of surface markers of mesenchymal stem cells in stock solution

umbilical cord source	Y200001	Y200003	Y210001
CD19	0.69%	0.26%	0.79%

CD34	0.34%	0.11%	0.79%
CD11b	0.15%	0.19%	0.84%
CD73	99.81%	99.95%	99.07%
CD90	99.81%	99.81%	99.99%
CD45	0.07%	0.23%	0.88%
CD105	98.45%	98.81%	99.12%
HLA-DR	0.10%	0.14%	0.35%

Example 2. Preparation of cryopreserved preparation of human umbilical cord-derived mesenchymal stem cells and its performance testing

1. Preparation of cryopreserved preparation of human umbilical cord-derived mesenchymal stem cells

1. Centrifuge the stock solution of mesenchymal stem cells prepared in Example 1 at 300 g for 8 minutes, discard all the supernatant after centrifugation, and collect the cell pellet. Then resuspend the cell pellet with the cell cryoprotectant (cryogenic solution) and cell density grouping in Table 2, respectively.

Table 2. Cell cryoprotectant and cell density

2. After completing step 1, draw the well-mixed cell suspension of each group into a 50mL syringe, inject it into the cryopreservation bag, and fill each bag with 12mL. After bagging, the air in the freezer bag is evacuated, and heat sealing is performed at a place 0.5 cm from the tube near the freezer bag. Put the cryopreservation bag into the cryopreservation folder, and put it into the cryopreservation rack in the programmed cooling instrument (Thermo Fisher Scientific (China) Co., Ltd., model: 7453). Open the valve of the liquid nitrogen tank, set and confirm the following cooling program on the computer:

Step 1: Initial temperature to 4°C;

Step 2: 1°C/min down to -5°C;

Step 3: 20°C/min until the cavity temperature is -40°C;

Step 4: 10°C/min until the cavity temperature is -20°C;

Step 5: 2°C/min until the sample drops to -40°C;

Step 6: 10°C/min until the sample drops to -80°C;

Step 7: End.

Run the program to cool down.

3. After completing step 2, transfer the cryopreservation folder to a liquid nitrogen tank transfer tank for storage.

2. Cell viability detection of cryopreserved preparations of human umbilical cord-derived mesenchymal stem cells

Taking stem cell stock solutions from 3 different umbilical cord sources (numbers Y200001, Y200003, and Y210001 respectively) as examples, they were prepared into 12 groups of mesenchymal stem cell cryopreservation preparations according to the different cryopreservation solutions and densities listed in step 1, and cryopreserved One week later, take it out and resuscitate at 37°C, mix the cells in the preparation evenly, take 20 μL of the sample and mix it with 20 μL of AO/PI fluorescent dye, draw 20 μL into the Countstar counting plate, and use a fluorescence counter for analysis.

The result is shown in Figure 3. The results showed that there was no significant difference in the activity rate of the preparations in the 12 groups, and the average values were all higher than 80%. It shows that the cell viability after thawing is good after the cell preparations prepared by the four cryopreservation solutions according to the cryopreservation density of 2.5×10 ⁶ cells/mL to 1×10 ⁷ cells/mL.

3. Identification of surface markers of different cryopreserved preparations of human umbilical cord-derived mesenchymal stem cells

Taking stem cell stock solutions from 3 different umbilical cord sources (numbers Y200001, Y200003, and Y210001) as examples, prepare mesenchymal stem cell cryopreservation preparations according to the different cryopreservation solutions listed in step 1 at a density of 5E6/mL. After the cryopreserved cells were recovered, the cell concentration was adjusted to 2×10 ⁶ /mL. Take the flow tube and add Mouse IgG1-FITC, antiCD19-FITC, antiCD34-FITC, Mouse IgG1-PE, antiCD11b-PE, antiCD73-PE, antiCD90-PE, antiCD45-PE, antiCD105-PE, antiHLA-DR- PE. The amount of antibody added was 5 μL, and 100 μL of the cell suspension to be tested was added respectively, shaken and mixed, and incubated at room temperature in the dark for 20 minutes. Add 2mL 1×PBS to each tube for washing, and centrifuge at 1200rpm for 5min. Discard the supernatant, add 200 μL 1×PBS to resuspend, mix well, and use BD FACSCalibur flow cytometer to detect 10,000 cells per sample.

The results are shown in Table 3-Table 5. The results showed that the surface expression of CD73, CD90 and CD105 on the surface of the stem cells of the cryopreserved preparation prepared by the method of the present invention were all greater than 95%, which was positive; CD34, CD45, CD11b, CD19 and HLA-DR were all less than 2%, which was negative. After cryopreservation, the cell surface markers did not change, which was consistent with the characteristics of stem cell surface markers.

Table 3. Detection of cell surface markers after cryopreservation of mesenchymal stem cell cryopreservation preparations prepared from Y200001-derived umbilical cord

Table 4. Detection of cell surface markers after cryopreservation of mesenchymal stem cell cryopreservation preparations prepared from umbilical cord Y200003

Table 5. Detection of cell surface markers after cryopreservation of mesenchymal stem cell cryopreservation preparations prepared from Y210001-derived umbilical cord

4. Identification of paracrine capacity of different cryopreserved preparations of human umbilical cord-derived mesenchymal stem cells

Taking stem cell stock solutions from 3 different umbilical cord sources (numbers Y200001, Y200003, and Y210001) as examples, prepare mesenchymal stem cell cryopreservation preparations according to the different cryopreservation solutions listed in step 1 at a density of 5E6/mL. After thawing, the cryopreserved cells were inoculated at 20,000/cm ² in a well plate, and cultured in an incubator with 5% CO ₂ and 37°C. After culturing for 48 hours, centrifuge to collect the supernatant, and use ELISA kits to detect the contents of IL-6, HGF, MMP1 and MMP2 in the supernatant respectively.

The results are shown in Table 6. The results show that: different mesenchymal stem cell cryopreservation preparations prepared by the method of the present invention have the ability to secrete IL-6, HGF, MMP1 and MMP2, thereby performing immune regulation.

Table 6. The ability of different mesenchymal stem cell cryopreserved preparations to secrete IL-6, HGF, MMP1 and MMP2

5. Expression levels of active factor mRNA in different cryopreserved preparations of human umbilical cord-derived mesenchymal stem cells

Taking stem cell stock solutions from 3 different umbilical cord sources (numbers Y200001, Y200003, and Y210001) as examples, prepare mesenchymal stem cell cryopreservation preparations according to the different cryopreservation solutions listed in step 1 at a density of 5E6/mL. After thawing, cryopreserved cells were inoculated at 20000/cm ² in the well plate, respectively in serum-free complete medium (normal culture group) and serum-free complete medium (inflammation stimulation group) added with IFN-γ (final concentration 15ng/mL) ) for cultivation. After culturing for 24 h, total RNA was extracted using an RNA extraction kit (TaKaRa, product number: 9767), and then the obtained total RNA was reverse-transcribed into cDNA using a reverse transcription kit (Thermo Fisher, product number K1621). cDNA was used as template for QPCR. The primer sequences are as follows:

CXCL10-F: 5'-GGTGAGAAGAGATGTCTGAATCC-3';

CXCL10-R: 5'-GTCCATCCTTGGAAGCACTGCA-3';

CCL2-F: 5'-GCTGTAAGGACATCGCCTACCA-3';

CCL2-R: 5'-AGAATCACCAGCAGCAAGTGTCC-3';

IDO-F: 5'-GCCTGATCTCATAGAGTCTGGC-3';

IDO-R: 5'-TGCATCCCAGAACTAGACGTGC-3'.

The QPCR system is as follows: upstream primer 0.4 μL; downstream primer 0.4 μL; Premix Ex Taq polymerase 10 μL; cDNA 2 μL; double distilled water 7.2 μL.

The QPCR procedure is as follows:

1) 95°C for 30 seconds, 1 cycle;

2) 95°C for 5 seconds, 60°C for 30 seconds, 40 cycles;

3) 95°C for 5 seconds, 60°C for 1 minute, 95°C, 1 cycle;

4) 1 cycle at 50°C for 30 seconds.

The results are shown in Figures 4-6. The results showed that the inflammatory environment could induce significant up-regulation of the mRNA expression levels of functional factors CCL2, CXCL10 and IDO1 in MSCs. It shows that the cryopreserved preparation of mesenchymal stem cells prepared by the method of the present invention has good functions of immune regulation and promoting damage repair.

6. Safety testing of cryopreserved preparations of human umbilical cord-derived mesenchymal stem cells

1. Experimental materials

Test product C1: the mesenchymal stem cell cryopreservation preparation prepared according to the method in step 1, the cell cryoprotectant is C1, and the cell concentration is 1.25×10 ⁷ cells/mL.

The test product vehicle: cell cryoprotectant C1.

Experimental animals: 40 SPF-grade CD-1 mice aged 6-7 weeks, male and female, provided by Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., and raised in SPF-grade Zhaoyan (Suzhou) New Drug Research Center Co., Ltd. The animal room was used for safety testing experiments after adaptive feeding for 1 week.

2. Experimental method

The mice were randomly divided into 2 groups according to body weight, and the dosage of each group is shown in Table 7. After grouping, all animals were given a single tail vein injection of 0.4 mL of corresponding test product C1 or test product vehicle, and the acute toxic reaction was observed by the side of the cage for at least 4 hours after administration, and then body weight and food intake were measured every week. After 14 days, anatomy and gross observation were performed, and abnormal tissues were examined by histopathology.

Table 7. Experimental grouping and dosage

3. Experimental results

During the experiment, none of the animals were found dead or dying. 1 male animal in group C1 of the test product showed purplish red at the distal end of the tail 2-9 days after administration, which was considered to be caused by mechanical damage during the administration and had nothing to do with the test product; Scabs were seen on the tails at 7-14 days; scabs were seen on the scrotum of 3 male animals 7-14 days after administration. Since the above animals were kept in the same cage, and the symptoms of scabs appeared at the same time, it may be caused by animal fights. irrelevant to the test article.

During the experiment, compared with the vehicle control group, the body weight gain of the male animals in the test product C1 group decreased in the first week (P<0.05, vs the vehicle control group), and the abnormality recovered in the second week. See Table 8 and Table 9 for details.

Table 8. Body weight and weight gain of male mice in each group (g, n=10, Mean±SD)

Note: *P<0.05 compared with vehicle control group.

Table 9. Body weight and weight gain of female mice in each group (g, n=10, Mean±SD)

During the experiment, compared with the vehicle control group, the food intake of the female and male animals W1 in the test product C1 group decreased, which may be related to the test product, and the abnormality recovered in the second week. In addition, the food intake of male animals W2 also decreased slightly, so it is considered not to have toxicological significance. The results are shown in Table 10.

Table 10. Food intake of mice in each group (g/only/day, n=10, Mean±SD)

Note: *P<0.05 compared with vehicle control group.

All animals were euthanized at the end of the administration period (D15), and no gross lesions related to the test article were found, so no tissue preservation and microscopic observation were performed.

To sum up, the cryopreserved preparation of human umbilical cord-derived mesenchymal stem cells (test product C1) was administered to CD-1 mice at a single intravenous injection of 5.0×10 ⁶ cells/only, and no animals died, nor did the test product related toxic reactions. Under the conditions of this experiment, the maximum tolerated dose of the test product C1 for mice is 5.0×10 ⁶ cells/mouse.

Example 3, Application of cryopreserved preparation of human umbilical cord-derived mesenchymal stem cells in the treatment of novel coronavirus pneumonia (COVID-19)

1. Experimental materials

Test product C1: the mesenchymal stem cell cryopreservation preparation prepared according to the method in step 1 of Example 2, the cell cryoprotectant was C1, and the cell concentrations were 1×10 ⁵ cells/mL, 1×10 ⁶ cells/mL, respectively. mL, 1×10 ⁷ cells/mL.

The test product vehicle: cell cryoprotectant C1.

Experimental animals: 9 7- to 8-week-old SPF-grade female hACE2-KI/NIFDC humanized mice (hACE2 mice), provided by the China Institute for Food and Drug Control, and raised in P4, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. Press the IVC feeding room.

2. Experimental method

The test animals were stratified and randomly divided into 3 groups according to body weight, namely the model control group, the C1 low-dose group and the C1 high-dose group, with 3 animals in each group. See Table 11 for specific grouping information.

Table 11. Animal grouping and dose design

After grouping, all animals were lightly anesthetized with carbon dioxide, and 5×10 ⁵ PFU of SARS-CoV-2 (HRB26) (SARS-CoV-2 (HRB26) was administered according to 50 μL/intranasal drip in the literature "Wang J, Shuai L, Wang C,et al.Mouse-adapted SARS-CoV-2 replicates efficiently in the upper and lower respiratory tract of BALB/c and C57BL/6J mice[J].Protein & cell,2020,11(10):776-782. "Middle), the day of infection is 0dpi. The drug treatment was carried out at 1dpi and 4dpi respectively. Each drug group was given the corresponding dose of the test product C1 by tail vein injection according to Table 11, and the model control group was given the same amount of the test product vehicle.

The general conditions of the animals were observed every day during the dosing period. Animal body weight was measured once a day. All animals were euthanized at 5 dpi, and part of the lungs were taken for determination of lung viral load (RT-qPCR). The remaining lungs (with part of the bronchi) were fixed in 4% paraformaldehyde, and were observed pathologically by HE staining.

3. Experimental results

Animals in each group did not die before and after the challenge, and no adverse reactions related to the test product C1 were seen.

The body weight changes of the low and high dose groups of test product C1 were consistent with those of the model group, and both showed a downward trend at 2dpi; the body weight of the administration group at some time points was different from that of the model control group, which may be related to the differences in body weight of each group before the challenge. The test product C1 had no significant effect on the state, body weight and weight gain of the model mice (Table 12).

Table 12. Effect of test product C1 on weight gain of COVID-19 mice (n=3, Mean±SD)

Note: *P<0.05 compared with the model control group.

Compared with the model control group, there was no statistical difference in the lung viral load of the low-dose and high-dose groups of test product C1 (P>0.05), but one (2F02) in the low-dose group was found to have no detected lung viral load, and the high-dose group The group found that the viral load in the lungs of 2 mice (3F02 and 3F03) decreased by about (1-2log10(copies/g)) (Table 13), suggesting that the test product C1 has a certain reduction in the viral load in the lungs of the COVID-19 model mice effect.

Table 13. Effect of test product C1 on viral load in lung tissue of COVID-19 mice (n=3, Mean±SD)

Inflammatory infiltration around small blood vessels: the inflammatory infiltration around small blood vessels in the model control group is mild to moderate; the degree of inflammatory infiltration in the low-dose C1 group has a tendency to improve, ranging from mild to moderate; in the high-dose C1 group The inflammatory infiltration was obviously improved, and it was negative-slight; it suggested that the test product C1 could dose-dependently improve the inflammatory infiltration around the small blood vessels of the lung (Table 14).

Inflammatory infiltration around the bronchiole: the inflammatory infiltration around the bronchiole in the model control group was mild to moderate; the inflammatory infiltration in the low dose group of test product C1 had a tendency to improve, and was negative to mild; the inflammation in the high dose group of test product C1 Sexual infiltration was significantly improved, and it was negative-mild; suggesting that the test product C1 can dose-dependently improve the inflammatory infiltration around the bronchioles of the lung (Table 14).

Degeneration of bronchial epithelial cells: the degeneration of bronchiole epithelial cells in the model control group was mild-slight; the degeneration of epithelial cells in the low-dose C1 group was significantly improved, and it was negative-mild; the high-dose C1 group was mild-slight Prompt that test product C1 has obvious improvement effect on lung bronchiole epithelial cell degeneration (Table 14).

Table 14. Effect of test product C1 on lung histopathology in COVID-19 mice

In summary, the test product C1 has a certain improvement on the lung viral load and lung histopathology of COVID-19 mice, among which the improvement on the inflammatory infiltration around the small blood vessels and bronchioles is the most significant, and the effective dose is 1×10 ⁵ cells/pcs.

Example 4. Application of cryopreserved preparation of human umbilical cord-derived mesenchymal stem cells in the treatment of idiopathic pulmonary interstitial fibrosis

1. Experimental materials

Test product C1: the mesenchymal stem cell cryopreservation preparation prepared according to the method in step 1 of Example 2, the cell cryoprotectant was C1, and the cell concentrations were 1×10 ⁶ cells/mL, 3×10 ⁶ cells/mL, respectively. mL, 1×10 ⁷ cells/mL.

The test product vehicle: cell cryoprotectant C1.

Test animals: 82 SPF-grade male SD rats aged 7-10 weeks, provided by Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., and kept in the SPF-grade animal room of Zhaoyan (Suzhou) New Drug Research Center Co., Ltd.

2. Experimental method

The test animals were divided into normal control group (10 rats) and model group (72 rats) according to body weight. On day 1 (D1) and day 3 (D3) after grouping, the model group was given bleomycin by atomization in the airway. Pulmonary fibrosis (PF) model was established with BLM 2.5mg/kg (D1) and 1mg/kg (D3), and the normal control group was given sodium chloride injection. After the second modeling, the animals in the model group were randomly divided into model control group, C1 low-dose group, C1 medium-dose group, and C1 high-dose group according to body weight, with 10 animals in each group. See Table 15 for details.

Table 15. Animal grouping and dose design

C1 low-dose group, C1 middle-dose group, and C1 high-dose group were given different doses of test substances (see Table 15 for specific doses) on D4, D7, and D10 tail veins respectively, and the normal control group and model control group were respectively administered on D4, D7. , D10 tail vein administration test product vehicle (cell cryoprotectant C1).

After the animals were anesthetized on D28, lung compliance (Cdyn), airway resistance (RL), forced vital capacity (FVC) and other indicators were tested using a pulmonary function analysis system. Then the whole lung tissue was taken and weighed to calculate the lung index. After the lung tissue was weighed, the left lung tissue was taken for hydroxyproline (HYP), MMP-2, and TIMP-1 content detection. The remaining right lung tissue and bronchi were fixed for histopathological examination.

3. Experimental results

After BLM modeling, the lung weight and lung index of rats in the model control group were significantly increased (P<0.01), and each dose group of the test product could reduce the lung index of PF model rats (P<0.01) (Table 16).

Table 16, the influence of test article on the lung index of BLM-induced PF model rats (n=10, Mean ± SD)

Note: Compared with the model control group, **P<0.01.

After BLM modeling, the Cdyn and FVC of rats in the control group were significantly lower than those in the normal control group, and RL was significantly higher. Each dose group of C1 can significantly improve the FVC of model rats, wherein the low dose group Cdyn, RL, and the high dose group also significantly improves Cdyn; suggesting that test product C1 can improve the impaired pulmonary function caused by fibrosis (Table 17 ).

Table 17. Effect of test product C1 on lung function of PF model rats induced by BLM (n=10, Mean±SD)

Note: Compared with the model control group, **P<0.01, *P<0.05.

After BLM modeling, HYP, MMP-2, and TIMP-1 in the lung tissue of the control group were significantly higher than those of the normal control group. Each group of C1 can significantly reduce HYP and TIMP-1, and the low and middle dose groups also significantly inhibit the overexpression of MMP-2, suggesting that the test product C1 can inhibit the expression of pro-fibrosis factors, improve collagen deposition, and play an anti-PF role (Table 18).

Table 18. Effect of test product C1 on fibrosis-related indicators of BLM-induced PF model rats (n=10, Mean±SD)

Note: Compared with the model control group, **P<0.01, *P<0.05.

After BLM modeling, moderate to severe multifocal inflammatory cell infiltration occurred in the alveoli of the animals in the model control group, moderate to severe multifocal fibrosis in the interstitium/alveoli of the lungs, accompanied by different degrees of multifocal alveolar space expansion, Macrophage aggregation, hemorrhage/congestion, fibrinous exudate. C1 can dose-dependently improve pulmonary interstitial/tracheal/perivascular/alveolar inflammatory infiltration and fibrosis, and the high-dose group is the most significant (Table 19, Figure 7).

Table 19. Effects of test product C1 on lung histopathology of BLM-induced PF model rats (n=10)

Note: "-" no lesion, "+" slight; "++" mild; "+++" moderate; "++++" obvious; "+++++" severe.

In summary, the test product C1 can improve the collagen deposition in lung tissue by inhibiting the expression of pro-fibrosis factors, and can significantly improve the lung function damage, lung inflammation and fibrosis caused by BLM, suggesting that the test product C1 has a better effect. Good anti-pulmonary interstitial fibrosis effect, the effective dose in rats is 1×10 ⁶ cells/kg.

Example 5. Application of cryopreserved preparation of human umbilical cord-derived mesenchymal stem cells in the treatment of acute lung injury

1. Experimental materials

Test product C1: the mesenchymal stem cell cryopreservation preparation prepared according to the method in step 1 of Example 2, the cell cryoprotectant was C1, and the cell concentrations were 1×10 ⁶ cells/mL, 3×10 ⁶ cells/mL, respectively. mL, 1×10 ⁷ cells/mL.

The test product vehicle: cell cryoprotectant C1.

Test animals: 70 SPF-grade male SD rats aged 6-8 weeks, provided by Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., and kept in the SPF-grade animal room of Zhaoyan (Suzhou) New Drug Research Center Co., Ltd.

2. Experimental method

The 70 male rats that passed the quarantine were randomly divided into 5 groups according to body weight, see Table 20 for details.

Table 20. Animal grouping and dose design

On the 1st day (D1) and the 3rd day (D3) after grouping, the animals in the model control group and each dose group of C1 were given lipopolysaccharide (LPS) 5mg/kg (D1 ), 0.8mg/kg (D3) to establish an acute lung injury (ALI) model, and the normal control group was given sodium chloride injection. After 4-6 hours of LPS administration on D1 and D3, different doses of the test substance were given through the tail vein according to Table 20, and the normal control group and the model control group were given the same amount of the test substance vehicle.

After D4 rats were anesthetized, 0.2 mL of arterial blood was collected for blood gas analysis. Subsequently, 2 mL of alveolar lavage fluid was collected, centrifuged and resuspended in 1 mL of PBS to settle to the bottom, and white blood cells and differential counts were performed with an automatic hematology analyzer. The middle lobe tissue of the right lung of the animal was taken and weighed, then placed in an oven at 60°C for 72 hours and weighed again to calculate the wet/dry weight ratio. The remaining right lung tissue and bronchus were fixed in 10% neutral buffered formalin solution for pathological examination.

The experimental data are expressed as "mean ± standard deviation". Statistical software SPSS 13.0 and/or GraphPad Prism 5 were used to process the data, and P<0.05 was considered statistically significant.

3. Experimental results

C1 can dose-dependently reduce the lung wet-to-dry weight ratio of ALI rats, and there is a significant difference between the middle and high dose groups of C1 and the model control group; suggesting that the test product C1 can reduce the water content of the lungs and improve pulmonary edema (Table 21).

Table 21. Effect of test product C1 on lung water content of LPS-induced ALI model rats (n=10, Mean±SD)

group	drug dosage	lung wet-to-dry ratio
normal control group	--	4.33±0.19**
Model control group	--	5.17±0.22
C1 low dose group	1×10 6 cells/kg	4.95±0.29 P=0.07
C1 middle dose group	3×10 6 cells/kg	4.91±0.19*
C1 high dose group	1×10 7 cells/kg	4.64±0.18**

Note: Compared with the model control group, **P<0.01, *P<0.05.

Each group of C1 can significantly increase the PCO2 of the model control group rats, and the PO2 and sO2 of the low-dose group and the PO2 of the middle-dose group are also significantly higher than the model control group; suggesting that the test product C1 has a significant effect on ALI rats with acute respiratory distress syndrome (ARDS). ) improved (Table 22).

Table 22. Effect of test product C1 on LPS-induced ALI model rat arterial blood gas analysis results (n=10, Mean±SD)

Note: Compared with the model control group, **P<0.01, *P<0.05.

Each dose group of C1 significantly reduced the abnormal increase of total protein content, leukocyte and differential count in the BALF of ALI rats, suggesting that the test product C1 had a significant improvement effect on lung inflammation (Table 23).

Table 23. Effect of test product C1 on total protein, leukocyte and differential count in BALF of ALI model rats (n=10, Mean±SD)

Note: Compared with the model control group, **P<0.01, *P<0.05.

Interstitial/alveolar inflammatory cell infiltration, broncho-associated lymphoid tissue hyperplasia, and alveolar hemorrhage can be seen in model animals. Each dose group of C1 can improve the inflammatory infiltration and lymphoid tissue hyperplasia of LPS-induced ALI model rats, suggesting that the test Product C1 has a certain therapeutic effect on pneumonia (Table 24, Figure 8).

Table 24. Effects of test product C1 on lung histopathology in ALI model rats (n=10)

Note: The degree of lesion "-" means normal; "+" means mild; "++" means mild; "+++" means moderate.

In summary, the test product C1 can significantly improve the lung inflammation, pulmonary edema and respiratory distress caused by LPS, suggesting that the test product C1 has a good anti-ALI effect, and the effective dose in rats is 1×10 ⁶ cells/ kg.

Example 6. Application of cryopreserved preparation of human umbilical cord-derived mesenchymal stem cells in the treatment of liver fibrosis

1. Experimental materials

Test product: the mesenchymal stem cell cryopreservation preparation prepared according to the method in step 1 of Example 2, the cell cryoprotectant is C1, and the cell concentrations are 1×10 ⁶ cells/mL and 5×10 ⁶ cells/mL, respectively , 1.5×10 ⁷ cells/mL.

The test product vehicle: cell cryoprotectant C1.

Test animals: 20 6- to 8-week-old SPF-grade male SD rats, provided by the Experimental Animal Center of the Academy of Military Medical Sciences of the Chinese People's Liberation Army, and bred in the animal room of Heze Biotechnology Co., Ltd.

2. Experimental method

Rats qualified for quarantine were injected intraperitoneally with CCL ₄ twice a week for 8 consecutive weeks to establish the liver fibrosis model. After modeling, they were randomly divided into 4 groups, see Table 25 for details.

Table 25. Experimental grouping and dose design

After grouping, according to Table 25, a single tail vein injection of the corresponding dose of C1 was given, and the model control group was given equal PBS. One week later, all rats were given a modeling dose of CCL ₄ by intraperitoneal injection. At the second week after treatment, blood was collected, serum was separated, and four items of transaminase and liver fibrosis were detected. Then the animals were sacrificed, the liver was weighed, and the liver index was calculated. HE staining and Masson staining were used for liver histopathological detection.

3. Experimental results

The test product C1 has a certain tendency to reduce the liver weight and liver coefficient of the liver fibrosis model rats, but there is no statistical difference compared with the model control group (Table 26).

Table 26. The effect of the test product C1 on the liver coefficient of CCL _4- induced liver fibrosis model rats (n=5, Mean±SD)

group	drug dosage	Liver weight (g)	Liver coefficient (%)
Model control group	--	20.36±4.41	3.82±0.44
C1 low dose group	1×10 6 cells/pc	17.74±2.85	3.33±0.42
C1 middle dose group	5×10 6 cells/pc	18.63±3.89	3.70±0.60
C1 high dose group	1.5×10 7 cells/pc	19.60±2.71	3.42±0.25

Each dose group of C1 had a certain improvement on the AST of model rats, and the serum AST level of the middle dose group of C1 was significantly lower than that of the model control group, but there was no obvious improvement on ALT (Table 27).

Table 27. Effects of test product C1 on liver function in CCL _4- induced liver fibrosis model rats (n=5, Mean±SD)

group	dose	ALT	AST
Model control group	--	47.42±7.60	126.14±12.59
C1 low dose group	1×10 6 cells/pc	46.30±9.43	102.24±24.54 p=0.08
C1 middle dose group	5×10 6 cells/pc	38.70±9.95	81.43±25.92*
C1 high dose group	1.5×10 7 cells/pc	41.68±13.82	113.20±18.24

Note: *P<0.05 compared with the model control group.

Each dose group of C1 has some improvement on the four indexes of liver fibrosis, among which the low and high dose groups of C1 can significantly reduce the content of HA and CIV in model rats, and the middle dose group can significantly reduce the content of PCIII (Table 28).

Table 28. Effects of test product C1 on the four items of liver fibrosis in rats with liver fibrosis caused by CCL ₄ (n=5, Mean±SD)

Note: Compared with the model control group, **P<0.01, *P<0.05.

The liver structure of the model control rats was disordered, and there were mild fatty degeneration and thickened collagen fiber deposition; the collagen in the low-dose C1 group did not disappear significantly, and the liver structure and collagen deposition in the middle and high-dose groups were significantly improved.

In summary, the test product C1 has a certain improvement effect on abnormal liver function and liver fibrosis caused by CCL ₄ , and the effective dose of the rat is 5×10 ⁶ cells/rat.

Example 7. Application of cryopreserved preparation of human umbilical cord-derived mesenchymal stem cells in the treatment of Crohn's disease

1. Experimental materials

Test product C1: the mesenchymal stem cell cryopreservation preparation prepared according to the method in step 1 of Example 2, the cell cryoprotectant was C1, and the cell concentrations were 1×10 ⁶ cells/mL, 2×10 ⁶ cells/mL, respectively. mL, 1×10 ⁷ cells/mL, 2×10 ⁷ cells/mL.

The test product vehicle: cell cryoprotectant C1.

Test animals: 46 SPF-grade male SD rats aged 6-8 weeks, provided by Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., and kept in the SPF-grade animal room of Beijing Zhaoyan New Drug Research Center Co., Ltd.

2. Experimental method

After passing the quarantine, the rats were randomly divided into 6 groups according to body weight. See Table 29 for specific grouping information.

Table 29. Animal grouping and dose design

After grouping, the rats were administered dinitrobenzenesulfonic acid (DNBS, 3.0mL/kg) in the colon to establish the Crohn's disease model, and the corresponding doses of the recipients were administered according to Table 29 on the next day (D1) and the fifth day (D5) after the modeling. The test substance, the normal control group and the model control group were given the same amount of the test substance vehicle.

After the DNBS model was established, the body weight was weighed every day, and the stool properties and bloody stool were observed and recorded, and the disease activity index (DAI) was evaluated according to Table 30. DAI=(weight loss score+stool properties score+blood in stool score)/3. D6 All animals were euthanized, colons were collected and measured for length and ulceration area, and then colon tissues were fixed for histopathological examination.

Table 30. Criteria for Scoring Clinical Symptoms

score	weight loss	Stool properties	blood in stool
0	0	normal	Fecal occult blood negative
1	0-5%	mild loose stool	Fecal occult blood positive
2	5%-10%	loose stool	Positive fecal occult blood
3	10%-20%	watery stool	Mild blood in stool
4	>20%	severe diarrhea	severe blood in stool

3. Experimental results

After modeling, the weight gain rate of the animals decreased significantly. Compared with the normal control group, the body weight of other groups decreased significantly on D1-D6, and there was no statistical difference among other groups (Table 31).

Table 31. Effect of test product C1 on body weight of Crohn's disease model rats induced by DNBS (n=6-8, Mean±SD)

After modeling, the DAI scores in each group increased significantly, and then gradually decreased. The DAI scores in the high-dose intraperitoneal injection group and the low-dose intravenous injection group on D5 and D6 showed a significant decrease trend, and the high-dose intraperitoneal injection group on D5 decreased by 58.00 compared with the model control group. %, the D6 intravenous injection low-dose group decreased by 33.33% compared with the model control group (Table 32).

Table 32. Effect of test product C1 on DAI score of DNBS-induced Crohn's disease model rats (n=6-8, Mean±SD)

Compared with the normal control group, the colon length in each group was significantly reduced, and the ulcer area was increased. The high-dose C1 intraperitoneal injection group had a tendency to improve the ulcer area, but there was no significant improvement in the colon length (Table 33).

Table 33. Effect of test product C1 on colon length and ulcer area of Crohn's disease model rats induced by DNBS (n=6-8, Mean±SD)

Gross necropsy of the euthanized animals on D6 showed melanosis in different areas of the colon in the model control group and C1 groups, and the incidence rate in the model control group was 7/8; the incidence rate in the C1 low-dose intraperitoneal group was 5/8; 2/6; the incidence rate of C1 intravenous low-dose group was 3/7; the incidence rate of C1 intravenous high-dose group was 3/7; each administration group had a certain reduction in the incidence of gross ulcer.

Under the microscope, colonic mucosal ulcers in the model control group and C1 administration groups were seen, mainly manifested as mucosal epithelial necrosis and exudation of neutrophils in the whole layer of the intestinal wall (mucosa, submucosa, muscular layer, adventitia), and granulation. Tissue formation; mucosal hemorrhage and crypt abscess can also be seen, but no significant improvement was seen in the test product C1.

In summary, intraperitoneal injection of the test product C1 ( ¹⁰⁷ /cause, 2 times/week) and tail vein injection of the test product C1 ( ¹⁰⁶ /cause, 2 times/week) have no effect on colon injury caused by modeling. There is a certain easing trend.

industrial application

The present invention adopts a serum-free medium cell culture process in the working library and preparation preparation stages of mesenchymal stem cells, which avoids the possible exogenous virus contamination caused by animal-derived raw materials, and at the same time does not contain ingredients in the serum-free medium And the content is relatively clear, avoiding the impact of differences between serum batches on product quality, and further improving the safety and stability of stem cell preparations. In addition, in order to ensure the timeliness, availability, safety, and effectiveness of clinical use, the present invention adopts the method of cryopreservation, and develops the preparation into an "off-the-shelf" product prepared in advance. Freeze in preparation form. Compared with fresh preparations, frozen preparations are more convenient to store, and can be directly administered intravenously after resuscitation without changing the liquid, avoiding the possible contamination risk during dispensing, and more conducive to clinical use. In addition, the frozen preparation has a long shelf life, and multiple tests on the function and safety of the preparation can be completed during the shelf life, with lower safety risks, which can effectively improve clinical benefits.

Claims (20)

1. A preparation method of human umbilical cord-derived mesenchymal stem cells, comprising the steps of:

   (1) culturing isolated umbilical cord tissue pieces, and obtaining P0 generation mesenchymal stem cells when the cell fusion degree of the mesenchymal stem cells climbing out from the umbilical cord tissue pieces is 40%-60%;

   (2) After step (1) is completed, the P0 generation mesenchymal stem cells are subcultured until the cell fusion degree is 60%-80%, and the P1 generation mesenchymal stem cells are obtained;

   (3) After completing step (2), subculture the P1-generation mesenchymal stem cells until the cell confluence is 60%-80%, and obtain P2-generation mesenchymal stem cells;

   (4) After step (3) is completed, the P2 generation mesenchymal stem cells are cryopreserved to obtain frozen P2 generation seed bank cells;

   (5) After completing step (4), the frozen P2 generation seed bank cells are recovered and subcultured, and cultured until the cell confluence is 60%-80%, to obtain P3 generation mesenchymal stem cells;

   (6) After step (5) is completed, the P3 generation mesenchymal stem cells are subcultured until the cell confluence is 60%-80%, and the P4 generation mesenchymal stem cells are obtained;

   (7) After step (6) is completed, the P4 generation mesenchymal stem cells are cryopreserved to obtain frozen P4 generation working bank cells;

   (8) After completing step (7), the frozen P4 generation working bank cells are recovered and subcultured, and the P5 generation mesenchymal stem cells are obtained when the cell confluency is 60%-80%;

   (9) After step (8), the P5 passage mesenchymal stem cells are digested, washed and resuspended in sequence to obtain the human umbilical cord-derived mesenchymal stem cells.
2. The method according to claim 1, characterized in that: said step (1), step (2) and step (3) all use fetal bovine serum complete medium for cell culture.
3. The method according to claim 1 or 2, characterized in that: in the step (5), step (6), and step (8), all serum-free complete medium is used for cell culture.
4. The method according to any one of claims 1-3, characterized in that: in the step (4), the method for freezing the cells is to resuspend the cells with the freezing solution A to obtain a cell suspension; the freezing Stock solution A consists of fetal bovine serum and dimethyl sulfoxide.
5. The method according to claim 4, characterized in that: the concentration of the cell suspension is 3×10 ⁶ cells/mL.
6. The method according to claim 4, characterized in that: the volume ratio of the fetal bovine serum and the dimethyl sulfoxide is 9:1.
7. The method according to any one of claims 1-6, characterized in that: in the step (7), the method for freezing the cells is to resuspend the cells with freezing solution B to obtain a cell suspension; Stock solution B consists of serum-free medium base, dimethyl sulfoxide and human albumin solution.
8. The method according to claim 7, characterized in that: the concentration of the cell suspension is 8×10 ⁶ cells/mL.
9. The method according to claim 7, characterized in that: the volume ratio of the serum-free medium base, the dimethyl sulfoxide and the human albumin solution is 7:2:1.
10. The human umbilical cord-derived mesenchymal stem cells prepared according to the method of any one of claims 1-9.
11. The application of the human umbilical cord-derived mesenchymal stem cells described in claim 10 in the preparation of cryopreserved preparations of human umbilical cord-derived mesenchymal stem cells.
12. A preparation for cryopreservation of human umbilical cord-derived mesenchymal stem cells, comprising the human umbilical cord-derived mesenchymal stem cells of claim 10 and a cell cryoprotectant.
13. The human umbilical cord-derived mesenchymal stem cell cryopreservation preparation according to claim 12, characterized in that: the cell cryoprotectant is composed of compound electrolyte solution, dimethyl sulfoxide, dextran 40 sodium chloride injection and human blood white protein solution composition.
14. The human umbilical cord-derived mesenchymal stem cell cryopreservation preparation according to claim 13, characterized in that: the compound electrolyte solution, the dimethyl sulfoxide, the dextran 40 sodium chloride injection and the human blood The volume ratio of albumin solution is (70-80):(5-10):(5-10):(5-10) or 70:(5-10):(5-10):(5-10) Or 80: (5-10): (5-10): (5-10) or (70-80): 5: (5-10): (5-10) or (70-80): 10: ( 5-10): (5-10) or (70-80): (5-10): 5: (5-10) or (70-80): (5-10): 10: (5-10) Or (70-80):(5-10):(5-10):5 or (70-80):(5-10):(5-10):10.
15. The human umbilical cord-derived mesenchymal stem cell cryopreservation preparation according to any one of claims 12-14, characterized in that: the human umbilical cord-derived mesenchymal stem cells in the human umbilical cord-derived mesenchymal stem cell cryopreservation preparation The concentration is 2.5×10 ⁶ -1×10 ⁷ cells/mL.
16. The preparation method of the human umbilical cord-derived mesenchymal stem cell cryopreservation preparation described in any one of claims 12-15, comprising the steps of: resuspending the human umbilical cord-derived mesenchymal stem cell described in claim 10 with a cell cryoprotectant, and then The obtained cell suspension is subjected to cooling treatment to obtain the cryopreserved preparation of human umbilical cord-derived mesenchymal stem cells.
17. The human umbilical cord-derived mesenchymal stem cells described in claim 10 or the human umbilical cord-derived mesenchymal stem cell cryopreservation preparations described in any one of claims 12-15 or the human umbilical cord-derived mesenchymal stem cells prepared according to the method described in claim 16 The application of the cryopreserved preparation of mesenchymal stem cells in any of the following N1)-N10):

    N1) Preparation of products for the prevention and/or treatment of diseases caused by novel coronaviruses;

    N2) Preparation of products for preventing and/or treating idiopathic pulmonary interstitial fibrosis;

    N3) Preparation of products for preventing and/or treating acute lung injury;

    N4) Preparation of products for preventing and/or treating liver fibrosis;

    N5) Preparation of products for preventing and/or treating Crohn's disease;

    N6) Prevention and/or treatment of diseases caused by novel coronaviruses;

    N7) prevention and/or treatment of idiopathic pulmonary interstitial fibrosis;

    N8) prevention and/or treatment of acute lung injury;

    N9) prevention and/or treatment of liver fibrosis;

    N10) Prevention and/or treatment of Crohn's disease.
18. A product whose active ingredient is the human umbilical cord-derived mesenchymal stem cells described in claim 10 or the cryopreserved preparation of human umbilical cord-derived mesenchymal stem cells described in any one of claims 12-15 or according to the method described in claim 16 The prepared cryopreserved preparation of human umbilical cord-derived mesenchymal stem cells; the function of the product is any one of M1)-M5):

    M1) Prevention and/or treatment of diseases caused by novel coronavirus;

    M2) prevention and/or treatment of idiopathic pulmonary interstitial fibrosis;

    M3) prevention and/or treatment of acute lung injury;

    M4) prevention and/or treatment of liver fibrosis;

    M5) Prevention and/or treatment of Crohn's disease.
19. A method for treating a disease caused by a novel coronavirus or idiopathic pulmonary interstitial fibrosis or acute lung injury or liver fibrosis or Crohn's disease, comprising the steps of: giving a patient with a disease caused by a novel coronavirus or idiopathic Patients with pulmonary interstitial fibrosis or patients with acute lung injury or patients with liver fibrosis or patients with Crohn's disease administer the human umbilical cord-derived mesenchymal stem cells described in claim 10 or the human umbilical cord-derived mesenchymal stem cells described in any one of claims 12-15 The cryopreserved preparation of mesenchymal stem cells or the cryopreserved preparation of human umbilical cord-derived mesenchymal stem cells prepared according to the method of claim 16 allows the patient to be treated.
20. The application according to claim 17 or the product according to claim 18 or the method according to claim 19, wherein the novel coronavirus is SARS-CoV-2.

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